Insulin regulates whole body glucose homeostasis, in part, by regulating the recruitment of the GLUT4 glucose transporter from intracellular compartments to the plasma membrane of muscle and fat cells. Thus, understanding how insulin acutely regulates glucose transport into fat and muscle requires a detailed understanding of insulin regulated-membrane trafficking. Insulin does not properly regulate GLUT4 trafficking in individuals with Type 2 diabetes. The molecular defect(s) underlying this insensitivity to insulin are not known. In the past decade, the majority of studies have focused on characterizing the intracellular and exocytic trafficking pathway of GLUT4. However, GLUT4 expression on the plasma membrane is a balance of endocytosis and exocytosis; hence, rapid GLUT4 internalization has a significant role in determining the amount of GLUT4 on the cell surface. The long-term objective of this project is to better characterize the mechanism that regulates the endocytosis of GLUT4 in adipocytes. In this application, I propose a quantitative, comprehensive analysis of insulin-regulation of GLUT4 internalization, the results of which will significantly extend our understanding of GLUT4 translocation and insulin action at a molecular level. The project has three specific aims. 1) We will use quantitative fluorescence microscopy and quantitative biochemical methods to characterize the internalization of GLUT4 in basal and insulin-treated adipocytes. These results will provide the conceptual foundation for the studies in the other aims. 2) We will analyze the internalization kinetics of GLUT4 in cells in which insulin-signal transduction has been perturbed by the expression of dominant-interfering mutants or by small interfering RNA knockdown of proteins of the insulin-signal transduction pathway. These results will provide novel information on the mechanisms of insulin regulation of endocytosis. 3) We will map, in studies of GLUT4 mutants, the structural determinants that control internalization in basal and insulin-stimulated conditions. The results of these studies will provide a more complete molecular description of insulin regulation of GLUT4 internalization, and thereby provide the necessary framework for understanding insulin-regulation of endocytosis at a molecular level.